Methods of detecting donor-specific antibodies and systems for practicing the same

ABSTRACT

Provided are methods for determining the presence or absence of donor specific antibodies in a biological sample. The methods include mixing a cellular sample from a donor with a biological sample from a recipient under conditions sufficient for recipient immune antibodies, if present, to bind to donor cell surface antigen (Ag) to form an immune antibody-Ag complex, contacting the mixture with beads comprising an antibody that specifically binds the immune antibody-Ag complex (e.g., the Ag or immune antibody) on a surface thereof, adding under lysis conditions a detectably-labeled antibody that specifically binds the immune antibody-Ag complex bound to the beads, and detecting the presence or absence of the detectably-labeled antibody bound to the immune antibody-Ag complex to determine the presence or absence of donor specific antibodies in the biological sample from the recipient. Systems and kits for practicing the subject methods are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to the filing date of U.S. ProvisionalPatent Application Ser. No. 61/782,003, filed Mar. 14, 2013, thedisclosure of which is incorporated herein by reference in its entirety.

BACKGROUND

Over 100,000 solid organ transplants are performed annually worldwide,including tens of thousands performed annually in the United States.Despite significant improvements in immunosuppression andpost-transplant care, long term graft function is less than optimal. Inthe United States, adjusted 10 year allograft survival rates fordeceased and living donor kidney transplants are only about 40% and 60%,respectively. Early and late stage graft failure, secondary to antibodymediated rejection (AMR) is a significant cause of poor graft survival.

Antibodies to Human Leukocyte Antigens (HLA) are circulating antibodiespresent in the transplant candidate or recipient's blood which are theresult of an earlier sensitization event (blood transfusion, previoustransplant, or pregnancy). Donor specific antibodies (DSA) presentpre-transplant can cause hyper-acute rejection and immediate graft lossand are assessed by a pre-transplant crossmatch. In more recent years,the concept of monitoring for the post-transplant development ofclinically relevant antibodies directed against donor specific HLA classI and class II mismatches has been a significant area of interest withinthe transplant community. Whether detected pre- or post-transplant, thepresence of antibodies directed against antigens expressed on donororgans, when not treated clinically, results in an immune attack on thetransplanted organ, and increases risk of graft loss and/or rejection.DSA attacks, among others, the endothelium of the allograft, and canresult in subsequent biopsy proven AMR and acute injury requiringaugmented immunosuppression. The progression of DSA development and thecorresponding clinical events compound to damage the allograft,resulting in chronic changes over time that ultimately compromise graftfunction and survival.

Antibody mediated rejection can present as an early acute process,resulting from an anamnestic response or de novo antibody production, oras a late and chronic process due to de novo antibody production. In theacute phase, it is often preformed antibodies that cause earlyrejection, but de novo DSA can also develop in the early post-transplantperiod, resulting in acute rejection. Patients with preformed DSA are atsignificantly greater risk of having an acute AMR and have significantlylower graft survival.

Chronic rejection is one of the leading causes of death-censored graftloss. Repeated cycles of alloantibody-mediated injury and repair resultin distinct changes in the microvasculature of the allograft. Patientswith preformed DSA and those who develop de novo DSA are at an increasedrisk of having chronic rejection.

SUMMARY

Provided are methods for determining the presence or absence of donorspecific antibodies in a biological sample. The methods include forminga mixture by combining a cellular sample from a donor with a biologicalsample from a recipient under conditions sufficient for recipient immuneantibodies, if present, to bind to donor cell surface antigen (Ag) toform an immune antibody-Ag complex, contacting the mixture with beadscomprising an antibody that specifically binds the immune antibody-Agcomplex (e.g., the Ag or immune antibody) on a surface thereof, addingunder lysis conditions a detectably-labeled antibody that specificallybinds the immune antibody-Ag complex bound to the beads, and detectingthe presence or absence of the detectably-labeled antibody bound to theimmune antibody-Ag complex to determine the presence or absence of donorspecific antibodies in the biological sample from the recipient. Systemsand kits for practicing the subject methods are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be best understood from the following detaileddescription when read in conjunction with the accompanying drawings.Included in the drawings are the following figures:

FIG. 1 schematically illustrates a method for determining the presenceor absence of donor specific antibodies in a biological sample accordingto one embodiment of the present disclosure.

FIG. 2 schematically illustrates a method for determining the presenceor absence of donor specific antibodies in a biological sample accordingto a second embodiment of the present disclosure.

FIG. 3 shows results of a DSA-FXM experiment involving simultaneouscapture and labeling of DSAs. Four samples were tested by DSA-FXM usingHLA-Class I and Class II beads distinguished by the internalfluorescence ID of each bead. Increasing fluorescence (positive signal)due to HLA specific antibody is shown on the X axis. FIG. 3, panel A:both HLA-Class I (C-I) and Class II (C-II) donor specific antibody (DSA)were negative (CI−/CII−); FIG. 3, panel B: only C-II DSA was positive(CI−/CII+); FIG. 3, panel C: only C-I DSA was positive (CI+/CII−); andFIG. 3, panel D: both CI and C-II DSAs were positive (CI+/CII+).

FIG. 4 shows results of a DSA-FXM experiment involving sequentialcapture and labeling of DSAs. A negative AB serum (Sample A) and threepositive sera (Samples B, C and D) were tested by DSA-FXM. Sample A:both C-I and C-II DSA negative; Sample B: both C-I and C-II DSApositive; Sample C: only C-I DSA positive and C-II DSA negative; SampleD: only C-II DSA positive and C-I DSA negative.

FIG. 5 provides results of a DSA-FXM experiment. A pool of HLA-Abpositive sera (PPS) in different dilutions was tested against variouscell numbers by FXM, DSA-FXM, and LMX-IgG. The results show DSA-FXM isthe most sensitive method for detecting DSA and uses many fewer cells(e.g. DSA can be detected with as few as 25,000 cells) when comparedwith standard methods. LMX-IgG defines the HLA specificities containedin the PPS serum on a Luminex platform using single antigen beads andthe values shown are the mean fluorescence intensities (MFI). Valuesgreater than or equal to 1000 MFI are considered positive; valuesbetween 500-999 MFI are considered possible positives (equivocal).

FIG. 6 shows results of a DSA-FXM experiment. Twenty-three CAP (theCollege of American Pathologists) external proficiency samples weretested by DSA-FXM simultaneously with the blinded challenge and inparallel with the regular flow cytometry crossmatch (FXM) and standardLuminex antibody screening on single antigen beads (LMX-IgG). The donorspecific antibodies (DSAs) of HLA-class I (C-I) and/or HLA-II (C-II)were identified and most DSAs were further confirmed by LMX-IgG. Someextra DSA with low MCS were only detected with the more sensitiveDSA-FXM method. External proficiency samples are sera and cells withknown specificities. The specificities of the sera are blinded to theparticipants until all results are received from all participatingcenters.

FIG. 7 provides results of a DSA-FXM experiment. Seven HLA-DQ DSApositive samples were identified by LMX-IgG and confirmed by DSA-FXM.Historically, it has been impossible to detect all specific DSA to DQ byany kind of DSA assay involving cells or cell extracts.

FIG. 8 shows results of a DSA-FXM experiment. Six HLA-DP DSA positivesamples were identified by LMX-IgG and confirmed by DSA-FXM.

FIG. 9 provides results of a DSA-FXM experiment. Three HLA-C DSApositive samples were identified by LMX-IgG and confirmed by DSA-FXM.

FIG. 10 shows experimental results from a DSA-FXM testing procedure.Panel A: both HLA-Class I (C-I) and Class II (C-II) donor specificantibody (DSA) were negative (CI−/CII−); Panel B: only C-I DSA waspositive (CI+/CII−); Panel C: only C-II DSA was positive (CI-CII+); andPanel D: both CI and C-II DSAs were positive (CI+/CII+).

FIG. 11 provides results of 117 separate DSA-FXM tests with known classI and class II DSA reactivity or lack thereof, showing mutuallyexclusive patterns of reactivity correlated with the known DSA profiles.

FIG. 12 shows a sensitivity comparison of FXM, DSA-FXM, and LMX-IgGsingle antigen bead assay for a class I specific DSA (B7), showing thatDSA-FXM can detect specific HLA class I DSA on the target cell even whenthe other two tests are negative.

FIG. 13 shows a sensitivity comparison of FXM, DSA-FXM, and LMX-IgGsingle antigen bead assay for a class II specific DSA (DR4), showingthat DSA-FXM can detect specific HLA class II DSA on the target celleven when the other two tests are negative.

FIG. 14 Panels A and B show the Pearson correlation between DSA-FXM andLMX-IgG SAB results for 95 class I DSA (Panel A) and 100 class II DSA(Panel B) FXM comparisons. Panel C shows sensitivity and specificitypercentages for class I and II using IgG DSA as the standard. Panel Dshows a comparison of LMX-IgG SAB, LMX-C1q SAB, FXM, and DSA-FXM on 7serum samples (6 individuals). Discrepancies related to over-reactivityof the LMX-SAB. In conjunction with FIG. 15, results show that theLMX-IgG SAB give false positive reactions (i.e., DSA positive when bothFXM and DSA-FXM are negative). This contributes to the lower specificityshown in FIG. 15.

FIG. 15 shows a comparison of FXM and DSA-FXM on 15 patients, 12 of whomhad autoantibody by FXM to antigens of unknown specificity (lowerpanel). Four of these patients (P12-P-15) had autoantibody directed toHLA as determined by DSA-FXM (upper panel)

FIG. 16 shows as comparison of the ability of FXM and DSA-FXM todistinguish positive reactions due to DSA class (I and/or II). DSA-FXMheaders: CI beads detect all class I, CIIa detects DQ, CIIb detects allDR and DP but only some DQ. Shown are four different types of results.Cases 1 and 3 both have positive B cell FXMs, but Case 1 is due to classI alloantibody whereas Case 3 is due to Class II alloantibody. Cases 2and 4 both have positive T and B FXMs, but Case 2 is due toautoantibody, whereas Case 4 is due to class I alloantibody.

FIG. 17 Panel A shows DSA-FXM results for class I DSA on serum diluted1:2 in buffer and 1:2 in intravenous immunoglobulin (IVIG). IVIG is usedfor desensitization to HLA, to lower antibody. The usual FXM showsincreases in the IVIG treated sample compared to buffer (data not shown)due to the presence of the second step anti-IgG reagent and broadreactivity of the IVIG with unknown targets on the cell surface. TheDSA-FXM shows inhibition of the IVIG because the detection is specificto HLA. Thus the DSA-FXM reveals efficacy of treatment. Panel B showsDSA-FXM results for class II DSA on serum diluted 1:2 in buffer and 1:2in intravenous immunoglobulin (IVIG). IVIG is used for desensitizationto HLA, to lower antibody. The usual FXM shows increases in the IVIGtreated sample compared to buffer (data not shown) due to the presenceof the second step anti-IgG reagent and broad reactivity of the IVIGwith unknown targets on the cell surface. The DSA-FXM shows inhibitionof the IVIG because the detection is specific to HLA. Thus the DSA-FXMreveals efficacy of treatment.

FIG. 18 Panel A shows results of a positive DSA serum spiked with 5%IVIG and tested at different dilutions by DSA-FXM. Results showed thatIVIG had a dose-dependent inhibition on both HLA class I DSAs. Panel Bshows results of a positive DSA serum spiked with 5% IVIG and tested atdifferent dilutions by DSA-FXM. Results showed that IVIG had adose-dependent inhibition on both HLA class II DSAs.

FIG. 19 shows FXM and DSA-FXM results on serial samples from a kidneycandidate undergoing IVIG desensitization treatment to prospectivelylower/abrogate DSA to an identified potential living donor. FXM resultsshow increased MCS values due to IVIG and Rituxan (therapeuticanti-CD20, a marker of B cells) while DSA-FXM results show inhibition(efficacy) of the IVIG and MCS values in the range acceptable fortransplant even in the presence of the therapeutic antibodies.

DEFINITIONS

By “donor specific antibodies” or “DSAs” is meant antibodies present ina recipient that specifically bind to donor antigens (e.g., donor cellsurface antigens). The DSAs may be “pre-formed” (e.g., present in therecipient prior to receiving a transplant or transfusion from a donor)and/or de novo DSAs which are produced by the recipient in response tohaving been pregnant, or receiving a transplant or transfusion from oneor more donors. The DSAs can, in some cases, be autologous DSAs(autoantibodies) that bind to cell surface components of the recipient'sown cells. In certain aspects, the DSAs are complement-fixing antibodies(CFAbs). In certain aspects, the DSAs are against HLA antigens.

An “affinity reagent” of the subject invention has an analyte bindingdomain, moiety, or component that has a high binding affinity for atarget analyte. By high binding affinity is meant a binding affinity ofat least about 10⁻⁴ M, usually at least about 10⁻⁶ M or higher, e.g.,10⁻⁹M or higher. The affinity reagent may be any of a variety ofdifferent types of molecules, so long as it exhibits the requisitebinding affinity for the target protein when present as tagged affinityligand.

As such, the affinity reagent may be a small molecule or large moleculeligand. By small molecule ligand is meant a ligand ranging in size fromabout 50 to about 10,000 daltons, usually from about 50 to about 5,000daltons and more usually from about 100 to about 1000 daltons. By largemolecule is meant a ligand in size from about 10,000 daltons or greaterin molecular weight.

Of particular interest as large molecule affinity ligands areantibodies, as well as binding fragments and mimetics thereof. Whereantibodies are the affinity ligand, they may be derived from polyclonalcompositions, such that a heterogeneous population of antibodiesdiffering by specificity are each tagged with the same tag. As such, theaffinity ligand may be a monoclonal, oligoclonal, and/or polyclonalantibody. The affinity ligand may be an antibody binding fragment ormimetic, where these fragments and mimetics have the requisite bindingaffinity for the target protein. For example, antibody fragments, suchas Fv, (Fab′)₂, and Fab may be prepared by cleavage of the intactprotein, e.g. by protease or chemical cleavage. Also of interest arerecombinantly produced antibody fragments, such as single chainantibodies or scFvs, where such recombinantly produced antibodyfragments retain the binding characteristics of the above antibodies.Such recombinantly produced antibody fragments generally include atleast the VH and VL domains of the subject antibodies, so as to retainthe binding characteristics of the subject antibodies. Theserecombinantly produced antibody fragments or mimetics of the subjectinvention may be readily prepared using any convenient methodology, suchas the methodology disclosed in U.S. Pat. Nos. 5,851,829 and 5,965,371;the disclosures of which are herein incorporated by reference.

The above described antibodies, fragments and mimetics thereof may beobtained from commercial sources and/or prepared using any convenienttechnology, where methods of producing polyclonal antibodies,oligoclonal antibodies, monoclonal antibodies, fragments and mimeticsthereof, including recombinant derivatives thereof, are known to thoseof skill in the art.

By “epitope” is meant a site on an antigen to which specific B cellsand/or T cells respond. The term is also used interchangeably with“antigenic determinant” or “antigenic determinant site.” An epitope cancomprise 1 or more amino acids, such as three or more amino acids, in aspatial conformation unique to the epitope. An epitope may include from1-10 amino acids, such as from 1-5 amino acids, e.g., 1, 2, 3, 4, or 5amino acids. Methods of determining spatial conformation of amino acidsare known in the art and include, for example, X-ray crystallography and2-dimensional nuclear magnetic resonance. Furthermore, theidentification of epitopes in a given protein is readily accomplishedusing techniques well known in the art. See, e.g., Geysen et al., Proc.Natl. Acad. Sci. USA (1984) 81:3998-4002 (general method of rapidlysynthesizing peptides to determine the location of immunogenic epitopesin a given antigen); U.S. Pat. No. 4,708,871 (procedures for identifyingand chemically synthesizing epitopes of antigens); and Geysen et al.,Molecular Immunology (1986) 23:709-715 (technique for identifyingpeptides with high affinity for a given antibody). Antibodies thatrecognize the same epitope can be identified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen.

By “binds specifically” or “specifically binds” is meant high avidityand/or high affinity binding of an antibody to a specific antigen orepitope. Antibody binding to its epitope on a specific antigen is with agreater avidity and/or affinity than binding of the same antibody todifferent epitopes, particularly different epitopes that may be presentin molecules in association with, or in the same sample, as a specificantigen of interest. Complement fixing antibodies may, however, have thesame or similar avidity and/or affinity for various epitopes ondifferent antigens of interest. As such, “binds specifically” or“specifically binds” is not meant to preclude a given complement fixingantibody from binding to more than one antigen of interest. Antibodiesthat bind specifically to a polypeptide of interest may be capable ofbinding other polypeptides at a weak, yet detectable, level (e.g., 10%or less of the binding shown to the polypeptide of interest). Such weakbinding, or background binding, is readily discernible from the specificantibody binding to the polypeptide of interest, e.g., by use ofappropriate controls.

By “detectably-labeled” antibody is meant an antibody having an attacheddetectable label, where the antibody is capable of binding specificallyto another molecule, e.g., another antibody (such as an IgG antibody).The detectably-labeled antibody retains binding specificity. Thedetectable label may be attached by chemical conjugation, or where thelabel is a polypeptide, it could be attached by genetic engineeringtechniques. Methods for production of detectably-labeled antibodies arewell known in the art. Detectable labels may be selected from a varietyof such labels known in the art, including radioisotopes, chromophores,fluorophores, fluorochromes, enzymes (e.g., horseradish peroxidase),linker molecules or other moieties or compounds which either emit adetectable signal (e.g., radioactivity, fluorescence, color) or emit adetectable signal after exposure of the label to its substrate. Variousdetectable label/substrate pairs (e.g., horseradishperoxidase/diaminobenzidine, biotin/streptavidin, luciferase/luciferin),methods for labeling antibodies, and methods for using labeled secondaryantibodies to detect an antigen are well known in the art. See, e.g.,Harlow and Lane, eds. (Using Antibodies: A Laboratory Manual (1999) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

By “isolated” is meant a compound of interest that is in an environmentdifferent from that in which the compound naturally occurs. “Isolated”is meant to include compounds that are within samples that aresubstantially enriched for the compound of interest and/or in which thecompound of interest is partially or substantially purified. The term“isolated” encompasses instances in which compound is unaccompanied byat least some of the material with which it is normally associated inits natural state. For example, the term “isolated” with respect to apolypeptide generally refers to an amino acid molecule devoid, in wholeor part, of sequences normally associated with it in nature; or asequence, as it exists in nature, but having heterologous sequences inassociation therewith.

As used herein, “purified” means that the recited material comprises atleast about 75% by weight of the total protein, with at least about 80%being preferred, and at least about 90% being particularly preferred. Asused herein, the term “substantially pure” refers to a compound that isremoved from its natural environment and is at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which it is naturally associated.

A “biological sample from a recipient” as used herein refers to a sampleof tissue or fluid isolated from a recipient, which in the context ofthe invention generally refers to samples which may contain donorspecific antibodies, which samples, after optional processing, can beanalyzed in an in vitro assay. Samples of interest include, but are notlimited to, blood, plasma, serum, blood cells, urine, saliva, biopsytissue, and mucous. Samples also include samples of in vitro cellculture constituents including but not limited to conditioned mediaresulting from the growth of cells and tissues in culture medium, e.g.,recombinant cells, and cell components.

By “human leukocyte antigen” or “HLA” is meant the genes within themajor histocompatability complex (MHC), which spans approximately 3.5million base pairs on the short arm of chromosome 6. The MHC isdivisible into 3 separate regions which contain the class I, the classII and the class III genes. In humans, the class I HLA complex is about2000 kb long and contains about 20 loci. Within the class I region existgenes encoding the well characterized class I MHC molecules designatedHLA-A, HLA-B and HLA-C. In addition, there are non-classical class Igenes encoded by the HLA-E, HLA-F, HLA-G, HLA-H, HLA-J, HLA-X and MICloci. The class II region contains six gene families encoded by theHLA-DRB1,3,4,5, HLA-DQA, HLA-DQB, and HLA-DPA, HLA-DPB loci. These genesencode the α and β chain of the classical class II MHC moleculesdesignated HLA-DRB1, 3, 4, 5, DQ and DP. In humans, non-classical genesencoded by the DM, DN and DO loci have also been identified within classII. The class III region contains a heterogeneous collection of morethan 36 loci associated with the immune response.

The terms “determining”, “measuring”, “evaluating”, “assessing” and“assaying” are used interchangeably and include quantitative andqualitative determinations.

The term “solid substrate” refers to a solid support in which antigensand/or antibodies may be immobilized thereon. Exemplary solid substratesinclude multiwell plates, membranes including nitrocellulose membranesand polyethylene membranes, cell and cell membranes, beads,microparticles, microspheres, microbeads, and the like. The methods ofthe invention may be carried out with microparticles, microspheres,microbeads, or beads of any material, e.g. silica, gold, latex, polymerssuch as polystyrene, polysulfone, polyethyl, or hydrogel. In addition,the microparticles, microspheres, beads or microbeads may be a magnetic.

The term “complement fixing antibody” refers to an antibody that bindsspecifically to an antigen or a pathogen and initiates the complementcascade of the immune system that provides for clearance of the antigenbearing target (e.g., cell) or pathogen from the organism. In general, acomplement fixing antibody is an IgM or an IgG antibody that isrecognized and specifically bound by complement factor C1q, complementfactor C3 via the alternate pathway, or the like.

It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely”,“only” and the like in connection with the recitation of claim elements,or the use of a “negative” limitation.

DETAILED DESCRIPTION

Provided are methods for determining the presence or absence of donorspecific antibodies in a biological sample. The methods include forminga mixture by combining a cellular sample from a donor with a biologicalsample from a recipient under conditions sufficient for recipient immuneantibodies, if present, to bind to donor cell surface antigen (Ag) toform an immune antibody-Ag complex, contacting the mixture with beadscomprising an antibody that specifically binds the immune antibody-Agcomplex (e.g., the Ag or immune antibody) on a surface thereof, addingunder lysis conditions a detectably-labeled antibody that specificallybinds the immune antibody-Ag complex bound to the beads, and detectingthe presence or absence of the detectably-labeled antibody bound to theimmune antibody-Ag complex to determine the presence or absence of donorspecific antibodies in the biological sample from the recipient. Systemsand kits for practicing the subject methods are also provided.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andexemplary methods and materials may now be described. Any and allpublications mentioned herein are incorporated herein by reference todisclose and describe the methods and/or materials in connection withwhich the publications are cited. It is understood that the presentdisclosure supersedes any disclosure of an incorporated publication tothe extent there is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anelectrode” includes a plurality of such electrodes and reference to “thesignal” includes reference to one or more signals, and so forth.

It is further noted that the claims may be drafted to exclude anyelement which may be optional. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely”, “only” and the like in connection with the recitation of claimelements, or the use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.To the extent such publications may set out definitions of a term thatconflict with the explicit or implicit definition of the presentdisclosure, the definition of the present disclosure controls.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Methods

As summarized above, aspects of the invention include methods fordetermining the presence or absence of donor specific antibodies in abiological sample. The methods include forming a mixture by combining acellular sample from a donor with a biological sample from a recipientunder conditions sufficient for recipient immune antibodies, if present,to bind to donor cell surface antigen (Ag) to form an immune antibody-Agcomplex, contacting the mixture with beads comprising an antibody thatspecifically binds the immune antibody-Ag complex (e.g., the Ag orimmune antibody) on a surface thereof, adding under lysis conditions adetectably-labeled antibody that specifically binds the immuneantibody-Ag complex bound to the beads, and detecting the presence orabsence of the detectably-labeled antibody bound to the immuneantibody-Ag complex to determine the presence or absence of donorspecific antibodies in the biological sample from the recipient. Varioussteps and aspects of the methods will now be described in greater detailbelow.

By “donor” is meant a source (e.g., a human source) of the cellularsample. The donor may be different from the recipient (e.g., where theDSAs may be alloantibodies), or the donor and recipient may be the same(e.g., where the DSAs may be autoantibodies). In certain aspects, thedonor may be a candidate for donating cells (e.g., blood cells), tissues(e.g., cornea, skin, bone, heart valve, tendon, femoral and/or saphenousveins, lymph nodes, spleen, and the like), organs (e.g., a kidney,heart, liver, pancreas, lung, intestine, eye, and the like), and anycombinations thereof, to a recipient in need thereof. Donors of interestinclude human donors, non-human primate donors, mammalian donors (e.g.,pigs), non-mammalian donors, and any other donor types of interest.

As used herein, a “cellular sample” from a donor is a sample obtainedfrom the donor that includes at least one cell. The at least one cellmay be a nucleated cell (e.g., a lymphocyte or peripheral bloodmononuclear cell (PBMC)), or a cell lacking a nucleus (e.g., anerythrocyte or platelet). In certain aspects, the cellular sample is asample obtained from the donor that includes cells selected fromlymphocytes (e.g., T cells and/or B cells), PBMCs, erythrocytes,platelets, and any combination thereof. According to certainembodiments, the cellular sample from the donor is from a tissue of thedonor (e.g., lymph nodes, spleen, cornea, skin, bone, heart valve,tendon, femoral and/or saphenous veins, and the like), from an organ ofthe donor (e.g., a kidney, heart, pancreas, lung, liver, intestine, eye,and the like), or any combination of such tissues and/or organs. Thecellular sample may be subjected to a purification procedure prior touse in the methods of the present disclosure. For example, the cellularsample may be a substantially pure sample of lymphocytes, peripheralblood mononuclear cells (PBMCs), erythrocytes, and/or platelets, whichsample is free of components that may interfere with the mixing,contacting and/or detecting steps of the subject methods. In certainaspects, the subject methods include obtaining the cellular sample fromthe donor.

According to certain embodiments, the cellular sample from the donorincludes 0.001×10⁶ to 2.0×10⁶ cells. In certain aspects, the cellularsample from the donor includes 1×10⁶ or fewer cells, such as 0.5×10⁶ orfewer cells, 0.4×10⁶ or fewer cells, 0.3×10⁶ or fewer cells, 0.2×10⁶ orfewer cells, 0.1×10⁶ or fewer cells, or 0.5×10⁵ or fewer cells. Incertain aspects, the cellular sample from the donor includes from 25,000to 200,000 cells.

By “recipient” is meant a source of the biological sample. The recipientmay be different from the donor (e.g., where the DSAs may bealloantibodies), or the recipient and donor may be the same (e.g., wherethe DSAs may be autoantibodies). In certain aspects, the recipient(e.g., a human recipient) may be a candidate for receiving cells (e.g.,blood cells), tissues (e.g., cornea, skin, bone, heart valve, tendon,femoral and/or saphenous veins, and the like), organs (e.g., a kidney,heart, pancreas, lung, liver, intestine, eye, and the like), and anycombinations thereof, from the donor (e.g., to alleviate a medicalcondition) or may have already received cells, a tissue, or an organfrom the donor. Recipients of interest include human recipients,non-human primate recipients, mammalian recipients, non-mammalianrecipients, and any other recipient types of interest.

The “biological sample” from the recipient may be any biological samplefrom the recipient which includes or may include donor specificantibodies (DSAs). According to certain embodiments, the biologicalsample from the recipient is selected from serum, plasma, blood, saliva,tissue, and any combination thereof. In certain aspects, the biologicalsample is 100 μL or less of serum, plasma, blood, saliva, or anycombination thereof, such as 90 μL or less, 80 μL or less, 70 μL orless, 60 μL or less, 50 μL or less, 40 μL or less, 30 μL or less, 20 μLor less, or 10 μL or less of serum, plasma, blood, saliva, or anycombination thereof. According to one embodiment, the biological samplefrom the recipient is 30 μL or less of serum, plasma, blood, saliva, orany combination thereof. In certain aspects, the subject methods includeobtaining the biological sample from the recipient.

Forming a mixture by combining a cellular sample from a donor with abiological sample from a recipient occurs under conditions sufficientfor recipient immune antibodies (e.g., DSAs), if present, to bind todonor cell surface antigen (Ag) to form an immune antibody-Ag complex.According to certain embodiments, the recipient immune antibodies arealloantibodies. In other aspects, the recipient immune antibodies areautoantibodies. Conditions sufficient for recipient immune antibodies(e.g., DSAs), if present, to bind to donor cell surface antigen (Ag) maybe provided by selection of a suitable buffer (e.g., PBS, TBS, or thelike), detergents (e.g., Tween), protein (e.g., BSA), pH, temperature,duration and/or the like. Conditions useful to permit specific bindingof antibodies to their target antigens are described, e.g., in Coligan,et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc.,NY (1994-2013). In certain aspects, the cellular sample from the donor(e.g., 0.2×10⁶ cells) and the biological sample from the recipient(e.g., 50 μL serum from the recipient) are incubated for about 20minutes at room temperature in a suitable buffer to permit the recipientimmune antibodies to bind to donor cell surface antigen. In certainaspects, the mixing, contacting and/or detecting steps are performed atroom temperature.

In certain aspects, the donor specific antibody is actual donor specificantibody.

According to certain embodiments, the donor cell surface antigen is anHLA antigen, such that the donor specific antibodies are anti-HLAantibodies. In certain aspects, the donor specific antibodies areanti-HLA class I and/or anti-HLA class II antibodies. According tocertain embodiments, the donor specific antibodies bind to a donor cellsurface antigen selected from HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3,HLA-DRB4, HLA-DRB5, HLA-DQA, HLA-DQB, HLA-DPA, and HLA-DPB.

The donor specific antibodies, if present, may be complement fixingantibodies (CFAbs). In certain aspects, methods of the invention includenot only determining the presence or absence of donor specificantibodies in the biological sample, but also include determiningwhether the DSAs are CFAbs or non-CFAbs. Identification of the DSAs asCFAbs may be performed, e.g., using a directly- or indirectly-labeledbinding agent that specifically binds to CFAbs (and/or non-CFAbs).According to certain embodiments, the binding agent is an isolatedcomplement component C1q. The C1q may be directly or indirectly labeledwith a fluorescent label that is distinguishable from any otherfluorescent labels in the complex. In certain aspects, the isolated C1qis conjugated to biotin (“Bio-C1q”) and may be detected by addition offluorescently-labeled streptavidin (e.g., R-phycoerythrin-conjugatedstreptavidin (SA-PE)). According to the above embodiments, the C1qprotein binds to the Ag-Ab DSA if the DSA is a complement fixingantibody, and the directly or indirectly labeled C1q may be detected inthe complex (along with the differentially detectably labeled antibodybound to the DSA) during the downstream detection step of the method(e.g., in a flow cytometer), indicating that the DSA is a CFAb.

Following forming the mixture, the mixture is contacted with beads thatinclude an antibody that specifically binds the donor cell surfaceantigen (e.g., an HLA antigen) of the antibody-Ag complex. Depending onthe donor cell surface antigen of interest, such beads may becommercially available. Otherwise, the desired type of bead (e.g., anagarose, latex, polystyrene, magnetic, or other type of bead) may beconjugated to an antibody that binds the antigen of interest usingconjugation strategies known in the art. See, e.g., G. T. Hermanson,“Bioconjugate Techniques” Academic Press, 2nd Ed., 2008. Moreover, kitsincluding reagents and instructions for conjugating an antibody ofinterest to a bead are commercially available (e.g., the Dynabeads®Antibody Coupling Kit (Life Technologies, Carlsbad, Calif.)). The beadsmay be microbeads having an average bead diameter of from 0.1 to 20microns, such as from 0.5 to 10 microns, e.g., 5 microns or less (e.g.,2.5 to 5 microns). The contacting is typically carried out underconditions sufficient for the antibody included on the bead tospecifically bind the donor cell surface antigen (e.g., an HLA antigen)to which the recipient immune antibody (e.g., a DSA) is already bound.Providing such conditions may include selection of a suitable buffer(e.g., PBS, TBS, or the like), detergent (e.g., Tween), protein (e.g.,BSA), pH, temperature, duration and/or the like. Conditions useful topermit specific binding of antibodies to their target antigens aredescribed, e.g., in Coligan, et al., eds., Current Protocols inImmunology, John Wiley & Sons, Inc., NY (1994-2013). Optionally, themixture is washed between the mixing and contacting steps.

The contacting step results in the immune antibody-Ag complex includingthe donor cell surface antigen bound by: a recipient immune antibody(e.g., a DSA), if present; and the antibody present on the bead. A donorcell of the cellular sample is also associated with this complex byvirtue of the donor cell surface antigen of the complex remaining on thesurface of the donor cell. Following the contacting step, adetectably-labeled antibody that specifically binds the complex (e.g.,the recipient immune antibody (e.g., the DSA) of the complex) is addedunder lysis conditions. Optionally, one or more wash steps are performedbetween the contacting step and the addition of the detectably labeledantibody under lysis conditions. The lysis conditions are sufficient tolyse the cells associated with the complexes, thereby freeing thecomplexes from the donor cells and facilitating downstream analysis ofthe complexes (e.g., by flow cytometry). In certain aspects, the lysisconditions include administering a lysis buffer that includes tracer,detergent, protease inhibitor, and BSA.

The detectably-labeled antibody and the lysis conditions may be providedby adding a “lysis mix” to the mixture after the contacting step, wherethe lysis mix includes the detectably labeled antibody in a lysisbuffer. Any suitable lysis buffer may be used and may include one ormore of Tris-HCl, EDTA, EGTA, SDS, deoxycholate, Triton X, NP-40, and/orany other desirable lysis buffer components. The lysis buffer is suchthat the immune antibody-Ag complex remains intact. The lysis buffer isnon-denaturing according to certain aspects of the present disclosure.The immune antibody-Ag complex now includes the bead-associated antibodybound to the donor cell surface antigen, the recipient immune antibody(e.g., DSA), if present, bound to the donor cell surface antigen, andthe detectably labeled antibody bound to the recipient immune antibody(e.g., DSA), if present. The detectable signals from the detectablylabeled antibody of the complex may be measured and proportionallycorrelated with the amount of recipient immune antibody (e.g., DSA) inthe biological sample from the recipient.

As set forth above, the methods of the present disclosure includedetecting (e.g., quantitatively detecting) the presence or absence ofthe detectably-labeled antibody bound to the immune antibody-Ag complexto determine the presence or absence of donor specific antibodies in thebiological sample from the recipient. The detection strategy employedmay vary according to the types of detectable label(s) present on thedetectably labeled antibody. Detectable labels that find use inpracticing the subject methods include, but are not limited to, afluorophore, a chromophore, an enzyme, a linker molecule, a biotinmolecule, an electron donor, an electron acceptor, a dye, a metal, or aradionuclide.

According to certain embodiments, the detectably labeled antibody isfluorescently-labeled and includes a fluorophore selected fromindocarbocyanine (C3), indodicarbocyanine (C5), Cy3, Cy3.5, Cy5, Cy5.5,Cy7, Texas Red, Pacific Blue, Oregon Green 488, Alexa fluor-355, AlexaFluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor-555, AlexaFluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, AlexaFluor 680, Alexa Fluor 700, JOE, Lissamine, Rhodamine Green, BODIPY,fluorescein isothiocyanate (FITC), carboxy-fluorescein (FAM),Allophycocyanin (APC), phycoerythrin (PE), rhodamine, dichlororhodamine(dRhodamine), carboxy tetramethylrhodamine (TAMRA), carboxy-X-rhodamine(ROX), LIZ, VIC, NED, PET, SYBR, PicoGreen, and RiboGreen.

When the detectably labeled antibody is fluorescently-labeled, thedetecting may include detecting one or more fluorescence emissions. Thefluorescence emission(s) may be detected in any useful format. Incertain aspects, the detecting includes flowing the immune antibody-Agcomplexes (which include a bead) through a flow cytometer.

When the detecting includes flowing the recipient immune antibody-Agcomplexes through a flow cytometer, the flow cytometer is configured todetect and uniquely identify the complexes by exposing the complexes toexcitation light and measuring the fluorescence of each complex in oneor more detection channels, as desired. The excitation light may be fromone or more light sources and may be either narrow or broadband.Examples of excitation light sources include lasers, light emittingdiodes, and arc lamps. Fluorescence emitted in detection channels usedto identify the complexes may be measured following excitation with asingle light source, or may be measured separately following excitationwith distinct light sources. In certain aspects, the flow cytometerthrough which the mixture is flowed includes fluorescence excitation anddetection capabilities such that the fluorescent label of the detectablylabeled antibody, and any other optional fluorescent labels associatedwith other components of the complex are each detectable anddistinguishable upon interrogation of the complexes by the flowcytometer.

Flow cytometers further include data acquisition, analysis and recordingmeans, such as a computer, where multiple data channels record data fromeach detector for the light scatter and fluorescence emitted by eachcomplex as it passes through the sensing region. The purpose of theanalysis system is to classify and count complexes where each complexpresents itself as a set of digitized parameter values. The flowcytometer may be set to trigger on a selected parameter in order todistinguish the complexes of interest from background and noise.“Trigger” refers to a preset threshold for detection of a parameter. Itis typically used as a means for detecting passage of a complex throughthe laser beam. Detection of an event which exceeds the threshold forthe selected parameter triggers acquisition of light scatter andfluorescence data for the complex. Data is not acquired for complexes orother components in the medium being assayed which cause a responsebelow the threshold. The trigger parameter may be the detection offorward scattered light caused by passage of a complex through the lightbeam. The flow cytometer then detects and collects the light scatter andfluorescence data for the complex.

Flow cytometric analysis of the complexes, as described above, yieldsqualitative and quantitative information about the complexes. Wheredesired, the above analysis yields counts of the complexes of interestin the mixture. As such, the flow cytometric analysis provides dataregarding the numbers of one or more different types of complexes in themixture.

The mixing, contacting and detecting steps may be performed collectivelyin any convenient amount of time. According to certain embodiments, themethods of the present disclosure are performed in 12 hours or less,such as 11 hours or less, 10 hours or less, 9 hours or less, 8 hours orless, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours orless, 3 hours or less, or 2 hours or less.

According to certain embodiments, methods of the present disclosureinclude generating a report indicating whether donor specific antibodiesare present in the biological sample from the recipient. If DSAs arepresent, the report may include information regarding the amount of DSAin the biological sample from the recipient. The report may be generatedby a computer, in which case the report is optionally displayed to anoutput device at a location remote to the computer.

In certain embodiments, methods of the present disclosure are used todetect DSAs that bind to donor HLA antigens (e.g., HLA class I and/orHLA class II antigens) present on cells in the cellular sample from thedonor. According to one embodiment, a mixture is formed by combining acellular sample that includes HLA antigen-containing donor cells withserum or plasma from the recipient such that any DSAs capable ofspecifically binding to the donor HLA may bind to the donor HLA. Theresultant mixture containing the DSA-HLA complexes may be washed one ormore times (e.g., three times) prior to the contacting step. Accordingto this embodiment, the contacting step includes adding anti-HLAantibody-coated microbeads such that the anti-HLA antibodies attached tothe beads bind to the constant regions of donor HLA class I and/or classII molecules on the surface of the donor cells. The resultant complex,which now includes capture beads bound to the donor HLA antigens, may bewashed one or more times (e.g., three times) before proceeding with themethod. According to this embodiment, fluorescently-labeled anti-IgGantibodies (e.g., PE-anti-IgG antibodies) are added under lysisconditions, such that the anti-IgG antibodies bind the DSA of thecomplex. Lysis of the donor cells facilitates separation of thecomplexes from other material present in the mixture. Next, fluorescencefrom the fluorescently-labeled anti-IgG antibodies may be detected, andoptionally quantitated, to determine the presence (and optionally theamount/concentration) or absence of DSAs in the recipient serum orplasma which bind to donor HLAs. In certain aspects, the methods areused to interrogate the recipient serum or plasma for the presence orabsence of DSAs that bind to HLA Class I and/or Class II, e.g., HLA-A,HLA-B, HLA-C, HLA-HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA,HLA-DQB, HLA-DPA, and HLA-DPB.

A method according to one embodiment of the present disclosure isschematically illustrated in FIG. 1. According to this embodiment,immune antibody-Ag complexes are detected by a flow cytometer or Luminexmachine. In the reaction, donor cells are combined with recipient serum.Donor specific antibody (DSA), if present, specifically binds to theantigen (Ag) on donor cells to form a DSA-Ag complex. Under lysisconditions, the DSA-Ag complex is specifically captured by beadsconjugated with antibody against the same Ag as in the DSA-Ag complex.The captured DSA-Ag is detected by a fluorescently-labeled secondaryantibody through a flow cytometer or Luminex machine.

A method according to a second embodiment of the present disclosure isschematically illustrated in FIG. 2. According to this embodiment,immune antibody-Ag complexes are detected by an enzyme-linkedimmunosorbent assay (ELISA). In the reaction, donor cells are combinedwith recipient serum. Donor specific antibody (DSA), if present,specifically binds to the antigen (Ag) on donor cells to form a DSA-Agcomplex. Upon lysis of the cell, the DSA-Ag complex is specificallycaptured on a substrate via an antibody against the same Ag as in theDSA-Ag complex. An enzyme linked secondary anti-IgG antibody binds tothe DSA, and the DSA is detectable upon reaction of the enzyme andsubstrate. Variations of this approach (e.g., luminescence assays) arealso provided by the present disclosure.

Systems

Also provided are systems for performing the methods of the presentdisclosure. Systems of the present disclosure include a sample fluidsubsystem that includes a processor and a computer-readable mediumoperably coupled to the processor with stored programming thereon. Whenexecuted by the processor, the stored programming programs the processorto form a mixture by combining a cellular sample from a donor with abiological sample from a recipient under conditions sufficient forrecipient immune antibodies, if present, to bind to donor cell surfaceantigen (Ag) to form an immune antibody-Ag complex. When executed by theprocessor, the stored programming also programs the processor to contactthe mixture with beads comprising an antibody that specifically bindsthe immune antibody-Ag complex on a surface thereof, and add under lysisconditions a detectably labeled antibody that specifically binds theimmune antibody-Ag complex. The subject systems also include a flowcytometer configured to assay the sample for the presence or absence ofthe detectably labeled antibody bound to the immune antibody-Ag complexto determine the presence or absence of donor specific antibodies. Incertain aspects, the flow cytometer is fluidically coupled to the samplefluidic subsystem.

The processor may be any suitable processor for executing the storedprogramming. According to certain embodiments, the processor isprogrammed to cause the sample fluidic subsystem to wash the mixturebefore the subsystem contacts the mixture with the with beads comprisingan antibody that specifically binds the immune antibody-Ag complex on asurface thereof. Alternatively, or additionally, the processor may beprogrammed to cause the sample fluidic subsystem to wash the mixtureafter the subsystem contacts the mixture with the beads comprising anantibody that specifically binds the immune antibody-Ag complex, butbefore the flow cytometer assays the sample for the presence or absenceof detectable labels bound to the complexes.

The computer readable medium may be a computer readable signal medium ora computer readable storage medium. A computer readable storage mediummay be, for example, but not limited to, an electronic, magnetic,optical, electromagnetic, infrared, or semiconductor system, apparatus,or device, or any suitable combination of the foregoing. More specificexamples (a non-exhaustive list) of the computer readable storage mediumwould include the following: an electrical connection having one or morewires, a portable computer diskette, a hard disk, a random access memory(RAM), a read-only memory (ROM), an erasable programmable read-onlymemory (EPROM or Flash memory), an optical fiber, a portable compactdisc read-only memory (CD-ROM), an optical storage device, a magneticstorage device, or any suitable combination of the foregoing. A computerreadable storage medium may be any tangible medium that can contain, orstore a program for use by or in connection with the sample fluidicsubsystem of the systems of the present disclosure.

The cellular sample from the donor, the donor cell surface antigens, thebiological sample from the recipient, the recipient immune antibodies(e.g., anti-HLA DSAs), the beads comprising an antibody thatspecifically binds the immune antibody-Ag complex on a surface thereof,the detectably labeled antibodies, the buffers, binding and lysisconditions, and the flow cytometer may be as described hereinabove withrespect to the methods of the present disclosure.

The systems of the present disclosure may be configured to detect thepresence or absence of DSAs in a convenient amount of time. According tocertain embodiments, the subject systems are configured to detect thepresence or absence of DSAs in a biological sample of the recipient in12 hours or less, such as 11 hours or less, 10 hours or less, 9 hours orless, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours orless, 4 hours or less, 3 hours or less, or 2 hours or less.

Kits

Kits which include one or more reagents useful for performing themethods of the present disclosure are also provided. According to oneembodiment, provided is a kit that includes a plurality of beadscomprising antibodies that specifically bind an immune antibody-Agcomplex on a surface thereof, a detectably labeled antibody thatspecifically binds the immune antibody-Ag complex, and instructions forusing the plurality of beads and the detectably labeled antibody toassay a cellular sample from a donor and a biological sample from arecipient to determine the presence or absence of donor specificantibodies in the biological sample. The subject kits may furtherinclude other useful components such as lysis buffer, control serum orplasma, a control cellular sample, and the like. The beads comprising anantibody that specifically binds an immune antibody-Ag complex on asurface thereof, and the detectably labeled antibody that specificallybinds the immune antibody-Ag complex, may be as described hereinabovewith respect to the methods of the present disclosure.

Reagents included in the subject kits may be provided in separate tubes,or two or more reagents may be provided in a single tube. According toone embodiment, the beads and detectably labeled antibodies are providedin separate tubes. In certain aspects, the detectably labeled antibodyis provided in a lysis buffer.

According to one embodiment, instructions included in the subject kitsare provided on a computer-readable medium which, when executed by aprocessor, programs the processor to assay a cellular sample from adonor and a biological sample from a recipient to determine the presenceor absence of DSAs in the biological sample.

Utility

The subject methods, systems and kits find use in any application inwhich it is desirable to detect donor specific antibodies in abiological sample of a recipient. Recipients of interest include, butare not limited to, human recipients in need of, or having alreadyreceived, an organ (e.g., kidney, liver, heart, etc.) or tissuetransplant from an organ or tissue donor. Applications of interestinclude pre-transplantation risk assessment and/or post-transplantationmonitoring based on detecting and/or quantifying the levels of DSAs inthe biological sample of the recipient.

The methods of the present disclosure allow the distinction betweenantibodies reactive to the donor cells and antibodies reactive to theHLA molecules on the donor cells. Prior flow crossmatch technologies aredeficient in that they are not capable of making this importantdistinction, where a positive flow crossmatch result might be completelyirrelevant to antibody interactions with HLA.

The subject methods provide a platform which can be broadly used todevelop many different DSA assays. Any type of biological sample fromthe recipient and any target cell of interest may be used to determinewhether DSA is present or absent. Compared to existing approaches, thesubject methods allow recipient antibodies to bind to donor antigens intheir native configuration without any modifications and with highdetection specificity and high throughput. Moreover, the methods may becompleted in less time, and require fewer donor cells, than existing DSAdetection approaches. Background signal caused by antibodies unrelatedto the target antigen of interest is eliminated by virtue of specificantibody-mediated solid-phase capture of complexes that include DSAsbound to the antigen of interest.

EXAMPLES

As can be appreciated from the disclosure provided above, the presentdisclosure has a wide variety of applications. Accordingly, thefollowing examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Those of skill in the art will readily recognizea variety of noncritical parameters that could be changed or modified toyield essentially similar results. Efforts have been made to ensureaccuracy with respect to numbers used (e.g. amounts, temperature, etc.)but some experimental errors and deviations should be accounted for.

Example 1: DSA-FM Testing Procedure

The following procedure may be used to practice one embodiment of thesubject methods, termed donor specific antibody flow cytometriccrossmatch (“DSA-FXM”). This example procedure involves simultaneouscapture and labeling, and can be completed in 2 hours or less.

First, prepare a 96-well layout format to arrange the FXM samples to betested. Second, dispense 0.2×10⁶ (less than 250 μl in volume) donorcells in all pre-selected wells in a 96-well plate according the platelayout. Centrifuge at 2,000×g for 3 minutes. Flick the plate and blottwice on a stacked paper tower before turning the plate over. Resuspendthe cells by gentle vortexing. Add 50 μl/well of each serum to thepre-selected wells according the plate layout. Gently vortex the plateand incubate in a RT incubator (22° C.) for 20 minutes. Prepare lysismix (for each well, PE-anti-hIgG and lysis buffer in a total volume of23 μl) during the incubation. After the incubation, add 250 μl of 3%HBSA to each well and spin the plate as before. Flick the plate and blottwice on a stacked paper tower before turning the plate over. Repeat thewash steps for an additional 2 times by adding 250 μl 3% HBSA for eachwash.

Add 5 μl Capture Beads Mix to each well at the last wash (3^(rd) wash),and wash again as before using 250 μl 3% HBSA. Add 23 μl lysis mix (celllysis buffer and fluorescence antibody) to each well and gently vortexthe plate. Cover the plate with a piece of foil and incubate the platein the dark with gentle shaking for 30 minutes. Prepare DSA-FXM washbuffer during the incubation as follows: to make 10 ml DSA-FXM washbuffer, add 0.5 ml detergent to 9.5 ml 1× TBS wash buffer and mix byinverting the tube five times.

Wash twice by adding 250 μl DSA-FXM wash buffer to each well and washingas before. Add 250 μl DSA-FXM wash buffer and wash as before. Resuspendthe beads in each well with 200 μl Flow Fixative and gently vortex theplate. Place the plate onto a flow cytometer and acquire the beads.Experimental results are shown in FIG. 3, FIGS. 5-9, FIGS. 11-14, andFIG. 16.

For the experiment shown in FIG. 3, four samples were tested by DSA-FXM(the simultaneous capture and labeling embodiment) and HLA-Class I andClass II beads were distinguished by the fluorescence ID on each bead.Increasing fluorescence (positive signal) due to HLA specific antibodyis shown on the X axis (FL1 channel). FIG. 3, panel A: both HLA-Class I(C-I) and Class II (C-II) donor specific antibody (DSA) were negative(CI−/CII−); FIG. 3, panel B: only C-II DSA was positive (CI−/CII+); FIG.3, panel C: only C-I DSA was positive (CI+/CII−); and FIG. 3, panel D:both CI and C-II DSAs were positive (CI+/CII+).

As shown in FIG. 5, a pool of HLA-Ab positive sera (PPS) in differentdilutions was tested against various cell numbers by FXM, DSA-FXM, andLMX-IgG. The results show DSA-FXM is the most sensitive method fordetecting DSA and uses many fewer cells (e.g. DSA can be detected withas few as 25,000 cells) when compared with standard methods. LMX-IgGdefines the HLA specificities contained in the PPS serum on a Luminexplatform using single antigen beads and the values shown are the meanfluorescence intensities (MFI).

As shown in FIG. 6, 23 external proficiency CAP samples (the College ofAmerican Pathologists) were tested by DSA-FXM simultaneously with theblinded challenge and in parallel with the regular flow crossmatch (FXM)and standard Luminex antibody screening on single antigen beads(LMX-IgG). The donor specific antibodies (DSAs) of HLA-class I (C-I)and/or HLA-II (C-II) were identified and most DSAs were furtherconfirmed by LMX-IgG. Some extra DSA with low MCS were only detectedwith the more sensitive DSA-FXM method. External proficiency samples aresera and cells with known specificities. The specificities of the seraare blinded to the participants until all results are received from allparticipating centers. The data presented in FIG. 7 indicates that sevenHLA-DQ DSA positive samples were identified by LMX-IgG and confirmed byDSA-FXM. As shown in FIG. 8, six HLA-DP DSA positive samples wereidentified by LMX-IgG and confirmed by DSA-FXM. As shown in FIG. 9,three HLA-C DSA positive samples were identified by LMX-IgG andconfirmed by DSA-FXM.

As shown in FIG. 11, a collection of 117 sera including HLA typingreagents, negative controls, and clinical samples were tested whose DSAspecificities were known from LMX-IgG SAB testing. Exclusive positive ornegative reactions were obtained. When the results of the DSA-FXM werecompared to the FXM and LMX-IgG SAB results, DSA-FXM had superiorsensitivity for class I (FIG. 12) and class II (FIG. 13) than either ofthe other tests. FIG. 14, Panels A and B gives the overall correlationfor 95 class I and 100 class II DSAs, respectively. FIG. 14, Panel Csummarizes the sensitivity and specificity of the DSA-FXM compared tothe LMX-IgG SAB assay. As shown in FIG. 14, Panel D, the reducedspecificity obtained in the FIG. 14, Panel C comparison is due to falsepositive reactions in the LMX-IgG SAB assay and not to false negativereactions in the DSA-FXM assay.

As shown in FIG. 15, the T and B cell FXM assays yield non-specific(i.e., not due to HLA, target unknown) positive results in the presenceof autoantibodies, whereas the DSA-FXM clearly distinguishesautoantibodies to HLA and discriminates whether the autoantibodies areto class I or class II.

As shown in FIG. 16, the DSA-FXM is able to distinguish flow cytometryresults due to class I and/or class II alloantibody as well as toautoantibody which the current FXM method in general use cannot do.Similar results seen with the FXM (e.g., Cases 1 and 3 or 2 and 4) havecompletely different explanations and interpretations when tested byDSA-FXM. The DSA-FXM can be correlated with the specific class I and/orII DSA profiles obtained by LMX-IgG SAB to give a prognosis for risk ofrejection pre- or post-transplant, whereas the FXM results cannot.

As shown in FIG. 17 for class I (Panel A) and class II (Panel B), theDSA-FXM is able to show inhibition of class specific DSA by IVIGtreatment as compared to buffer. This parallels what is seen in vivobefore and after IVIG infusion.

As shown in FIG. 18 for class I (Panel A) and class II (panel B), DSAspecific sera show a dose-dependent inhibition by IVIG which alsopredicts in vivo efficacy.

As shown in FIG. 1, FXM and DSA-FXM were performed using serial samplesfrom a kidney candidate undergoing IVIG desensitization treatment toprospectively lower/abrogate DSA to an identified potential livingdonor. FXM results show increased MCS values (i.e., became morepositive) due to an artifact of IVIG infusion. The artifact is due tothe second step antibody (anti-human IgG) which is used as the signal inthe assay. Because all of the IVIG product is purified IgG, FXM resultsshow false positive increases due to the IVIG, not to the DSA. Rituxan(therapeutic anti-CD20, a marker of B cells) also increases MCS valuesin the B cell FXM because of the CD20 on the B cell surface. Althoughthis is not artifact, the FXM is designed to detect HLA antibody, notnative cell specific targets. DSA-FXM results, in contrast, showinhibition (efficacy) of the IVIG and MCS values in the range acceptablefor transplant even in the presence of the therapeutic (anti-CD20)antibodies.

Result Calculations

Use a DSA-FXM Analysis Worksheet to record and perform the calculations.Determine the Median Channel Shift (MCS) for Patient Sera & Positivecontrols. For calculation of MCS: MCS=Median Channel Value (MCV) of thepatient sera (or Pos controls) minus Median Channel Value (MCV) of theNeg controls. Record the result as MCS on the worksheet and computer.Make a report according to the FXM cutoff to define a Negative orPositive DSA-FXM.

Results and Interpretation

The cutoffs for FXM were determined by the results (MCS) from pre-testedAB male sera (usually about 20) against 5 different sources of targetcells (fresh/frozen PBMC, frozen lymph node, and frozen spleen cells).MCS for each tested AB serum was calculated by subtracting negativecontrol MCV from AB serum MCV; and means of DSA-FXM MCS were calculatedfrom all MCSs obtained (N=138). The criteria of DSA-FXM cutoffs were setas follows: MCS values<AB neg MCS+3 SD were interpreted as “Negative”;MCS values>=AB neg MCS+3SD were interpreted as “Positive”. HLA-I DSA-FXMPositive: MCS>=61. HLA-II DSA-FXM Positive: MCS >=60.

Materials and Methods

For the FXM procedure, distribute 0.1×10⁶ PBMC cells into each well in a96-well plate and centrifuge at 1,300×g for 3 minutes. Flick the plateto remove the supernatant; resuspend the cells in 50 μl test serum andincubate in a RT incubator (22° C.) for 20 minutes. After theincubation, wash the cells four times with 250 μl of 3% HBSA each time.Add 100 μl detecting reagent mix containing 0.5 μg of FITC-anti humanIgG (Jackson ImmunoResearch Laboratories, Inc.; West Grove, Pa., USA),0.2 μg PerCP-CD3 and PE-CD19 (BD Biosciences, San Jose, Calif., USA).After an additional 30 minute incubation at room temperature, wash cellstwice with 250 μl of 3% HBSA each time and resuspend in 200 μl 0.2%paraformaldehyde in PBS. Acquire cells on BD FACSCanto II Flow Cytometer(BD Biosciences, San Jose, Calif., USA). The acquired data are analyzedby BD FACSDiva™ software.

For the LMX-IgG procedure, experiments were performed according to themanufacturer's instructions (One Lambda, Canoga Park, Calif., USA). Theresult shows that both HLA class I and II DSA are detected by DSA-FXM invarious conditions (FIG. 5).

Example 2: DSA-FXM Testing

The following procedure may be alternatively used to practice oneembodiment of the subject methods, termed donor specific antibody flowcytometric crossmatch (“DSA-FXM”). This example procedure involvessequential capture and labeling.

First, prepare a 96-well layout format to arrange the FXM setting.Second, dispense 0.2×10⁶ (less than 250 μl in volume) donor cells in allpre-selected wells in a 96-well plate according the plate layout.Centrifuge at 2,000×g for 3 minutes. Flick the plate to removesupernatant. Gently vortex the plate and add 50 μl/well of each serum tothe pre-selected wells according the plate layout. Gently vortex theplate and incubate in a RT incubator (22° C.) for 20 minutes. After theincubation, add 250 μl of 3% HBSA to each well and spin the plate asbefore. Flick the plate and blot twice on a stacked paper tower beforeturning the plate over. Repeat the wash steps for an additional 2 timesby adding 250 μl 3% HBSA for each wash.

Add 5 μl Capture Beads Mix containing HLA-class I and II capture beadsto each well at the last wash (3rd wash), and wash again as before using250 μl 3% HBSA. Add 23 μl cell lysis buffer to each well and gentlyvortex the plate. Cover the plate with a piece of foil and incubate theplate in the dark with gentle shaking for 30 minutes. Wash the beadstwice with 250 μl each DSA-FXM wash buffer as before. Resuspend thebeads with 100 ul of Fluorescent anti-IgG in wash buffer and incubatethe plate at RT for an additional 30 minutes. Wash the beads twice with250 μl each DSA-FXM wash buffer as before and resuspend the beads ineach well with 200 μl Flow Fixative and gently vortex the plate. Placethe plate onto a flow cytometer and acquire the beads. FXM and LMX-IgGprocedures were carried out as described above. The experimental resultsare shown in FIG. 4. As shown in FIG. 4, A negative AB serum (Sample A)and three positive sera (Samples B, C and D) were tested by DSA-FXM(sequential capture and labeling). Sample A: both C-I and C-II DSAnegative; Sample B: both C-I and C-II DSA positive; Sample C: only C-IDSA positive and C-II DSA negative; Sample D: only C-II DSA positive andC-I DSA negative.

Example 3: DSA-FXM Testing

The following procedure may be alternatively used to practice oneembodiment of the subject methods, termed donor specific antibody flowcytometric crossmatch (“DSA-FXM”). This example procedure involvessequential capture and labeling.

First, prepare a 96-well layout format to arrange the FXM setting.Second, dispense 0.2×10⁶ (less than 250 μl in volume) donor cells in allpre-selected wells in a 96-well plate according the plate layout.Centrifuge at 2,000×g for 3 minutes. Flick the plate to removesupernatant. Gently vortex the plate and add 50 μl/well of each serum tothe pre-selected wells according the plate layout. Gently vortex theplate and incubate in a RT incubator (22° C.) for 20 minutes. After theincubation, add 250 μl of 3% HBSA to each well and spin the plate asbefore. Flick the plate and blot twice on a stacked paper tower beforeturning the plate over. Repeat the wash steps for an additional 2 timesby adding 250 μl 3% HBSA for each wash.

Add 100 μl of Fluorescent anti-IgG to resuspend the cells and cover theplate with a piece of foil; and incubate the plate in the dark withgentle shaking for 30 minutes. Wash the cells twice with 250 μl eachDSA-FXM wash buffer as before. Resuspend the cells in 25 μl lysis bufferand add 5 μl Capture Beads Mix containing HLA-class I and II capturebeads to each well. Incubate the plate at RT for an additional 30minutes. Wash the beads twice with 250 μl each DSA-FXM wash buffer asbefore and resuspend the beads in each well with 200 μl Flow Fixativeand gently vortex the plate. Place the plate onto a flow cytometer andacquire the beads. FXM and LMX-IgG procedures were carried out asdescribed above. The experimental results are shown in FIG. 10 and FIG.11: FIG. 10, Panel A: both HLA-Class I (C-I) and Class II (C-II) donorspecific antibody (DSA) were negative (CI−/CII−); FIG. 10, Panel B: onlyC-I DSA was positive (CI+/CII−); FIG. 10, Panel C: only C-II DSA waspositive (CI−/CII+); and FIG. 10, Panel D: both CI and C-II DSAs werepositive (CI+/CII+).

Example 4: Auto-DSA-FXM Testing

Autologous sera from 15 recipients were tested against the recipients'own PBMC cells by FXM in parallel with the DSA-FXM procedure describedin Example 1. The experimental results are shown in FIG. 15: of 15 autocrossmatches, 3 were negative and 4 were positive by both DSA-FXM andFXM; 8 were only positive by FXM and proved that the DSAs detected byFXM were not DSAs against HLA antigens.

Example 5: Intravenous Immunoglobulin (IVIG) Desensitization DSA-FXMTesting

The inhibition effect of IVIG on HLA-DSA was evaluated by DSA-FXMtesting procedure described in Example 1. Experimental results are shownin FIGS. 17-19.

For the experiment shown in FIG. 17, two HLA DSA positive sera werespiked with 5% IVIG and tested in vitro by DSA-FXM. The inhibitioneffect of IVIG on both HLA class I and II DSA was measurable.

As shown in FIG. 18, a positive DSA serum in different dilutions wasspiked with 5% IVIG and tested by DSA-FXM. The result showed that IVIGhad a dose-dependent inhibition on both HLA class I and II DSAs.

A series of samples from a kidney candidate under IVIG desensitizationtreatment were tested against a potential (but incompatible) livingdonor's cells by both DSA-FXM and FXM. As shown in FIG. 19, theinhibition effect of IVIG on HLA-DSA was observed by DSA-FXM but couldnot be seen by FXM. Rituxan (therapeutic anti-CD20) had no interferenceon the test results by DSA-FXM testing.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

What is claimed is:
 1. A method for determining the presence or absenceof donor specific antibodies (DSAs) in a biological sample, the methodcomprising: forming a mixture by combining a donor cellular sample witha biological sample from a recipient under conditions sufficient fordonor specific antibodies (DSAs), if present, to bind to donor cellsurface antigen (Ag) to form DSA-Ag complex comprising donor cells boundto DSAs via the donor cell surface Ag; contacting the mixture with beadscomprising monoclonal antibody immobilized on a surface of the beads,that specifically binds the donor cell surface Ag in the DSA-Ag complex;adding under lysis conditions, to the mixture contacted with the beads,a detectably-labeled antibody that specifically binds the DSAs in theDSA-Ag complex bound to the beads, wherein the lysis conditions aresufficient to lyse the donor cells associated with the DSA-Ag complex;and detecting the presence or absence of the detectably-labeled antibodybound to the DSAs in the DSA-Ag complex bound to the beads to determinethe presence or absence of the DSAs in the biological sample from therecipient.
 2. The method according to claim 1, wherein the detecting issemi-quantitative.
 3. The method according to claim 1, wherein theimmune antibody is an alloantibody.
 4. The method according to claim 1,wherein the immune antibody is an autoantibody.
 5. The method accordingto claim 1, wherein the immune antibody is a complement fixing antibody(CFAb).
 6. The method according to claim 1, wherein the detectingcomprises detecting a fluorescence emission.
 7. The method according toclaim 1, wherein the detecting comprises flowing the complex through aflow cytometer.
 8. The method according to claim 1, wherein thedetecting comprises detecting the complex by an enzyme-linkedimmunosorbent assay (ELISA).
 9. The method according to claim 1, whereinthe detectably-labeled antibody comprises a detectable label attached tothe antibody or an antigen binding fragment thereof.
 10. The methodaccording to claim 1, wherein the detectable label comprises afluorochrome, a chromophore, an enzyme, a linker molecule, a biotinmolecule, an electron donor, an electron acceptor, a dye, a metal, or aradionuclide.
 11. The method according to claim 1, wherein thedetectable label comprises a fluorophore selected from the groupconsisting of: indocarbocyanine (C3), indodicarbocyanine (C5), Cy3,Cy3.5, Cy5, Cy5.5, Cy7, Texas Red, Pacific Blue, Oregon Green 488, Alexafluor-355, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, AlexaFluor-555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, AlexaFluor 660, Alexa Fluor 680, Alexa Fluor 700, JOE, Lissamine, RhodamineGreen, BODIPY, fluorescein isothiocyanate (FITC), carboxy-fluorescein(FAM), Allophycocyanin (APC), phycoerythrin (PE), rhodamine,dichlororhodamine (dRhodamine), carboxy tetramethylrhodamine (TAMRA),carboxy-X-rhodamine (ROX), LIZ, VIC, NED, PET, SYBR, PicoGreen, andRiboGreen.
 12. The method according to claim 1, wherein the donorspecific antibody is an antibody produced by the recipient in responseto receiving a transplant or transfusion from one or more donors. 13.The method according to claim 1, wherein the Ag is a Human LeukocyteAntigen (HLA).
 14. The method according to claim 1, wherein thebiological sample comprises serum, blood, saliva, or plasma.
 15. Themethod according to claim 1, comprising obtaining the cellular samplefrom the donor prior to forming the mixture, wherein the cellular samplecomprises lymphocytes.
 16. The method according to claim 1, comprisingobtaining the biological sample from the recipient prior to forming themixture, wherein the biological sample comprises serum.
 17. The methodaccording to claim 1, wherein the cellular sample from the donorcomprises nucleated cells.
 18. The method according to claim 1, whereinthe cellular sample from the donor comprises from 0.001×10⁶ to 2.0×10⁶cells.
 19. The method according to claim 1, wherein the cellular samplefrom the donor comprises fewer than 0.2×10⁶ cells.
 20. The methodaccording to claim 1, wherein the cellular sample from the donorcomprises fewer than 0.1×10⁶ cells.
 21. The method according to claim 1,wherein the cellular sample from the donor comprises fewer than 0.5×10⁵cells.
 22. The method according to claim 1, wherein the cellular samplefrom the donor comprises about 25,000 to 200,000 cells.
 23. The methodaccording to claim 1, wherein the average bead diameter is from 0.1 to20 microns.
 24. The method according to claim 1, wherein the averagebead diameter is 5 microns or less.
 25. The method according to claim 1,wherein the average bead diameter is between 2.5 to 5 microns.
 26. Themethod according to claim 1, wherein the beads are agarose beads, latexbeads, magnetic beads, or polystyrene beads.
 27. The method according toclaim 1, wherein the method is performed in 12 hours or less.
 28. Themethod according to claim 1, wherein the method is performed in 8 hoursor less.
 29. The method according to claim 1, wherein the lysisconditions comprise administering a lysis buffer comprising tracer,detergent, and DNase.
 30. The method according to claim 1, comprisinggenerating a report indicating whether donor specific antibodies arepresent in the biological sample from the recipient.
 31. The methodaccording to claim 30, wherein generating the report is performed by acomputer.
 32. The method according to claim 31, wherein the report isdisplayed to an output device at a location remote to the computer. 33.The method according to claim 1, wherein the recipient is a candidatefor receiving a transplant or a transfusion from the donor.
 34. Themethod according to claim 33, wherein the biological sample is serum,blood, or plasma.